Dry reagent particle assay and device having multiple test zones and method therefor

ABSTRACT

The present invention relates to a dry reagent assay device having at least one test zone and at least one reference zone, which provides an internal mechanism for assuring correct and reliable assay procedures and reagent qualities. In one embodiment, the present invention relates to an assay device having, at least one test zone for detecting at least one analyte in a sample by reacting the sample with a labeled indicator reagent, and a reference zone for receiving, unreacted labeled indicator reagent.

RELATED APPLICATION

The subject matter of this application is related to a disposablesingle-use digital electronic, instrument that is entirelyself-contained, including all chemistry reagents, as disclosed in U.S.application Ser. No. 08/111,347 entitled “Novel Disposable ElectronicAssay Device” filed Aug. 24, 1993 by Michael P. Allen. The aboveapplication has the same assignee as the present invention and isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a dry reagent assay device having twoor more test zones, which provides an internal reference mechanism forassuring correct and reliable assay procedures and reagent qualities.

BACKGROUND OF THE INVENTION

Qualitative and quantitative self-tests have developed gradually overthe last half century. Non-instrumented tests have become commerciallyavailable using immunochemical reagents on a solid support fordiagnostic tests involving HCG, LH, FSH, CKMB, Staphylococcus, andRubella. Measurement of the hormone HCG to detect pregnancy was amongthe first of these tests to become commercially successful in the homemarket. The first home pregnancy test, the e.p.t.™, as introduced in1977 by Warner-Lambert. The e.p.t.™ used a solution phase chemicalreaction that formed a brown ring on the surface of the urine solutionin the presence of HCG. The 2 hour long protocol associated with thistest was sensitive to vibration and timing, causing false results.

Two additional test systems that appeared in the late 1980s were theLipoScan™ by Home Diagnostics Inc. and the Chemcard™ by Chematics Inc.Bath tests measure cholesterol in whole-blood using visual colorcomparison. Since visual color matching is subjective these tests do notachieve the quantitative performance necessary for cholesterol testing(Pradella et al, Clin. Chem. 36:1994-1995 (1990)).

For many analytes such as the markers for pregnancy and ovulation,qualitative or semi-quantitative tests are appropriate. There are,however, a variety of analytes that require accurate quantitation. Theseinclude glucose, cholesterol, HDL cholesterol, triglyceride, a varietyof therapeutic drugs such as theophylline, vitamin levels, and otherhealth indicators. Generally, their quantitation has been achievedthrough the use of an instrument. Although suitable for clinicalanalysis, these methods are generally undesirable for point-of-caretesting in physicians offices and in the home due to the expense of theinstrument.

Recently, a number of non-instrumented methods for measuring analytesuse instrument-free quantitation through the use of migration distance,rather than color matching, as the visual signal. In migration distanceassays, chemical/biochemical reactions occur as the analyte is wickedthrough a solid support. During wicking the analyte reacts with asignal-producing reagent and forms a visible signal along the support.The migration distance or the distance of signal border is related toanalyte concentration. The operator reads the height of the color barmuch the same way one reads a thermometer, and finds the concentrationfrom a calibrated scale.

There are a few migration-type assays commercially available. Theseinclude Environmental Test Systems' Quantab™, which measures chloride inswimming pools and during the mixing of concrete, Syva's AccuLevel® forthe measurement of therapeutic drugs, and ChemTrak's AccuMeter® formeasurement of cholesterol in whole blood. Other companies such asEnzymatics and Crystal Diagnostics have more recently announced theintroduction, of their Q.E.D.™ and Clinimeter™ technologies to measure,respectively, alcohol in saliva and cholesterol in blood. ActiMed™discloses a thermometer-type cholesterol assay device in Ertinghausen,U.S. Pat. No. 5,087,556 (1992).

Although these single use, thermometer-type, non-1-instrumentedquantitative devices and non-instrumented color comparison devices forqualitative measurement have shown adequate performance, they haveseveral problems associated with reliability and convenience. First, thecolors generated on these devices are not always uniform and sharp inthe case of migration type assays the border is often light in color,unclear and difficult to read. This translates directly into user errorssince the user must make a judgment related to the position of the colorband border. DT the case of non-instrumented pregnancy tests it issometimes difficult to visually interpret the intensity of the coloredspot (especially at HCG concentrations close to the cut-offsensitivity), and interpretation of the result is sometimes a problem.Anytime a non-technical operator is required to make a visual judgmentor interpretation, an error is possible, and sometimes, is unavoidable.

Second, the assay protocol for these tests is sometimes difficult andlengthy, taking 15 minutes to 1 hour to obtain a result. Third, thesetests often do not have sufficient procedural and reagent references toassure adequate test performance. Fourth and last, non-instrumenteddevices can only measure single endpoint type tests since enzyme ratesor ratiometric analysis of two analytes cannot be measured. Therefore,the menu of potential tests is limited.

As an example of the significance of the problems, a recent article inClinical Chemistry (Daviaud et al, Clin. Chem. 39 53-59 (1993))evaluated all 27 home use pregnancy tests sold in France. The authorsstate, “among the 478 positive, urine samples distributed, 230 werefalsely interpreted as negative”.

In the past, immunoassays were developed for the quantitative andqualitative, determination of a wide variety of compounds in alaboratory setting using detailed procedures and expensiveinstrumentation. Recent developments in immuno-diagnostics have resultedin a movement toward more simple approaches to the rapid analysis ofclinical samples. The development of solid phase bound reagents haseliminated the need for precipitation in the separation of boundreagents from free reagents. Further advancements in solid phaseimmunochemistry have resulted in non-instrumented dry reagent stripimmunoassays. This configuration allows for the visual qualitative orsemi-quantitative determination of analytes in patient samples withoutthe use of an instrument.

There are two basic types of non-instrumented immunoassayconfigurations. In the first type, or visual color zone type, a signalis generated at a specific zone on the strip where the signal indicatesthe presence of analyte; and the intensity of the signal indicates theconcentration of the analyte in the sample. This type of assay requiresvisual color interpretation either for the presence of color above athreshold, as in the case of a qualitative test, or the comparison ofthe color intensity to a color chart, as in the case of asemi-quantitative test. In the second type, the visual signal isproduced along the length of a bibulous assay strip. During wicking, theanalyte reacts with a signal-producing reagent and forms a visiblesignal along the support. The migration distance of the signal from theproximal end of the strip is a direct measure of analyte concentration.This type of non-instrumented migration height assay can achievequantitative results with reasonable performance as disclosed in Zuk etal, Clin. Chem. 31:1144-1150 (1985).

The color zone type of strip immunoassay is usually configured in threeways. First, a one site competitive immunoassay where labeled reagentand analyte compete for binding sites at a discrete zone along a stripwhere one member of the binding pair is immobilized. Second, a one siteinhibition immunoassay where labeled reagent binds substantially all ofthe sample analyte prior to contact the strip zone where the oppositemember of the binding pair is immobilized. Third, a two-site or“sandwich” immunoassay, where the sample analyte has at least twobinding sites.

The prior art discloses color one immunoassays in lateral flow andvertical flow configurations limited to the use of enzymatic signalgenerating systems. The use of lateral flow wicking strips has focusedin the area of enzyme detection in one-site competitive or two-sitesandwich configurations, and the use of particle detection has beenconfined largely to two-site sandwich immunoassays.

There are examples of methods developed where chemical or immunologicalreactions occurred along the length of a bibulous assay strip. In U.S.Pat. Nos. 4,094,841, 4,235,801 and 4,363,537 Deutsch and Mead disclose abibulous strip assay with discrete immunochemical reagent zones alongits length for conducting specific binding assays. Grubb and Gladd, U.S.Pat. No. 4,168,146, describe an enzyme immuno-chromatography assay on abibulous strip wherein a sample containing antigen is wicked through theassay strip, and the antigen in the sample binds to the immobilized,antibody and progressively fills the binding sites as a measure ofanalyte concentration. The antigen containing area is visualized bywetting the strip with an enzyme labeled antibody and developing colorwith a chromogenic substrate. David, et al., disclose U.S. Pat. No.4,376,110 that monoclonal antibodies with binding affinities of. 10⁸ orgreater can be used in forward, reverse and simultaneous two-sitesandwich immunoassays.

The lateral wicking immunoassays using colored particle detection fortwo-site configurations in the prior art are limited to visuallyinterpretation and usually provide, only qualitative, or at best,semi-quantitative results. The prior art fails to disclose coloredparticle detection in lateral wicking devices which use competitive orinhibition immunoassay configurations. Likewise, lateral wickingimmunoassay reagent strips designed for use in a quantitative instrumentread format are not disclosed in the prior art. Furthermore, themultiple test zone reagent strips of the prior art fail to providequality reference.

Thus, a need exists in the field of diagnostics for a wicking assaywhich it sufficiently accurate and reliable to permit point-of-care useby untrained individuals in locations such as the home, sites of medicalemergencies, or locations other than a clinic.

SUMMARY OF THE INVENTION

The present invention provides, a device for determining the presence ofat least one of a plurality of analytes in a sample. The device includesa test zone corresponding to each analyte selected for determining itspresence. Each test zone receives and contacts the sample and a labeledindicator reagent corresponding to the selected analyte with a test zonereagent corresponding to the selected analyte. The test zone reagentcorresponds to the selected analyte reacting in the presence of thesample and the labeled indicator reagent corresponds to the selectedanalyte to form a corresponding test zone reaction product and acorresponding test zone detectable response inversely related to theselected analyte level in the sample. The device also includes areference zone for receiving from each test zone the labeled indicatorreagent not reacted with its corresponding test zone reagent andcontacting each labeled indicator reagent with a corresponding referencezone reagent. Each reference zone reagent reacts in the presence of thecorresponding labeled indicator reagent to form a correspondingreference zone reaction product and a corresponding reference zonedetectable response related to each selected analyte level in the sampleand related to the corresponding test zone detectable response toestablish a substantially constant total detectable response for apre-determined range of each selected analyte. The device also includescleans for combining the detectable responses from the test zones todetermine the analyte level in the sample.

The present invention also includes a device for determining thepresence of an analyte in a sample. The device includes a first testzone for receiving and contacting the sample and a labeled indicatorreagent with a first reagent. The first reagent reacts in the presenceof the sample and labeled indicator reagent to form a first reactionproduct and a detectable response in the first test zone inverselyrelated to the analyte level in the sample. A second test zone receivesand contacts the labeled indicator reagent hot reacted with the firstreagent with a second, reagent. The second reagent reacts in thepresence of the labeled indicator reagent to form a second reactionproduct and a detectable response in the second test zone related to theanalyte level in the sample and related to the detectable response ofthe first test zone to establish a substantially constant totaldetectable response from the test zones for a pre-determined range ofthe analyte. The device also includes means or combining the detectableresponses from the test zones to determine the analyte level, in thesample.

The present invention also provides a device for determining thepresence of an analyte in a sample which includes a porous membercapable of being traversed by the sample. A first zone on the porousmember receives and contacts the sample with labeled indicator reagentdiffusively immobilized on the porous member. The labeled indicatorreagent reacts in the presence of the analyte to form a mixture. Asecond zone on the porous member receives and contacts the mixture witha first reagent non-diffusely immobilized on the porous material in thesecond zone. The first reagent reacts in the presence of the mixture toform a first reaction product and a detectable response in the secondzone inversely, related to the analyte level in the sample. A thirdzone, on the porous member receives and contacts the remaining mixturewith a second reagent non-diffusely immobilized on the porous materialin the third zone. The second reagent reacts in the presence of theremaining mixture to form a second reaction product and a detectableresponse in the third, zone related to the analyte level in the sample.The device also includes means for determining the analyte level in thesample from the detectable responses in the second and third zones.

A preferred embodiment of the present invention provides a device fordetermining the presence of an analyte in a sample which includes abibulous member capable of being traversed by the sample. A first zoneon the bibulous member receives and contacts the sample with aparticle-linked antigen diffusively immobilized on the bibulous member.The particle-linked antigen reacts in the presence of the analyte toform a mixture. A second zone on the bibulous member receives andcontacts the mixture with an antibody non-diffusely immobilized on thebibulous material in the second zone. The antibody is a specific bindingpartner to the particle-linked antigen and the analyte, the antibodyreacts in the presence of the mixture to bind the particle-linkedantigen and express a detectable response in the second zone inverselyrelated to the analyte level in the sample. A third zone on the bibulousmember receives and contacts the remaining mixture with an antibodynon-diffusely immobilized on the bibulous material in the third zone.The antibody is a first member of a specific binding pair capable ofbinding to a second member of the specific binding pair on theparticle-linked antigen. The second member of the specific binding pairis not a specific binding partner to the analyte. The antibody reacts inthe presence of the remaining mixture to bind with the remaining mixtureand express a detectable response in the third zone related to theanalyte level in the sample. The device also includes means fordetermining the analyte level in the sample from the detectableresponses in the second and third zones.

Another preferred embodiment of the present invention provides a devicefor determining, the presence of an analyte in a sample which includes abibulous member capable of being traversed by the sample. A first zoneon the bibulous member receives and contacts the sample with aparticle-linked antibody diffusively immobilized on the bibulous member.The particle-linked antibody reacts in the presence of the analyte toform a mixture. A second zone on the bibulous member receives andcontacts the mixture with an antigen non-diffusely immobilized on thebibulous material in the second zone. The antigen is a specific bindingpartner to the particle-linked antibody. The antigen reacts in thepresence of the particle-linked antibody to substantially bind theparticle-linked antibody and express a detectable response in thesecond, zone inversely related to the analyte level in the sample. Athird zone on the bibulous member receives and contacts the remainingmixture with an antibody non-diffusely immobilized on the bibulousmaterial in the third zone. The antibody is a first member of a specificbinding pair capable of binding to a second member of the specificbinding pair on the particle-linked antibody. The second member of thespecific binding pair is not a specific binding partner to the analyte.The antibody reacts in the presence of the remaining mixture to bindwith the particle-linked antibody and express a detectable response, inthe third zone related to the analyte level in the sample. The devicealso includes means for determining the analyte level in the sample fromthe detectable responses in the second and third zones.

Methods are also provided by the present invention for determining thepresence of an analyte in a test sample. One method comprising the stepsof: contacting the sample with a porous member having a plurality ofzones; transporting the sample sequentially across the plurality of,zones and contacting the sample to at least one reagent immobilized, ineach zone; detecting a response from the contact between the sample andthe reagent in at least two zones; and, determining the analyte level inthe sample by combining the response from at least two zones.

Another method provided by the present invention determines the level ofat least one analyte in a, sample. The method comprising the steps ofcontacting the sample with an end portion of a bibulous, strip having, aplurality of zones; wicking the sample to a labeled indicator reagentdiffusively immobilized on the bibulous strip; reacting the labeledindicator reagent in the presence of the analyte to form a mixture;wicking the mixture to a first reagent non-diffusely immobilized on thebibulous strip; reacting the first reagent in the presence of themixture to form a first reaction product and a detectable responseinversely related to the analyte level in the sample, wicking theremaining mixture to a second reagent non-diffusely immobilized on thebibulous strip; reacting the second reagent in the presence of theremaining mixture to form a second reaction product and a detectableresponse related to the analyte level in the sample, and, determiningthe analyte level in the sample from the detectable responses in thereacting steps with the first and second reagents.

Accordingly, it is an object of the present invention to provide awicking device which uses competitive and immunoassay configurations fortest results which are more accurate and reproducible than in the priorart.

It is further object of the present invention to provide a assay methodusing multiple test zones in a single assay to yield accuratequantitative results.

Another object of the present invention is to, provide an assay which,provides mean for quality reference using the signals combined frommultiple test zones.

A farther object of the invention is to provide a quantitative stripimmunoassay based on particle detection.

Other and further advantages, embodiments, variations and the like willbe apparent to those skilled in the art from the present specificationtaken with the accompanying drawings and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which comprise a portion of this disclosure:

FIG. 1 shows a top surface view of an embodiment baying typicalstructure with three reagent zones that can be used for quantitative andqualitative immunoassays;

FIG. 2 shows a top surface view of an embodiment having typicalstructure with four reagent zones that can be used for quantitative andqualitative immunoassays;

FIG. 3 shows an exploded lengthwise, cross section of the embodiment ofFIG. 1;

FIG. 4 shows an exploded lengthwise cross section of an embodimenthaving a typical structure with a samplepre-treatment/filtration/separation/blood separation device;

FIG. 5 shows an exploded lengthwiSe cross section of an embodimenthaving a typical structure with a samplepre-treatment/filtration/separation/blood separation device and a sampletransport;

FIG. 6 shows the top surface view of one embodiment of the N-telopeptide(NTx) assay strip;

FIG. 7 shows the top surface view of a second embodiment of theN-telopeptide (NTx) assay strip; and

FIG. 8 is a graphical representation of a dose response to NTx withquality reference. Plotting reflectance density vs. NTx concentration.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns a lateral flow immunoassay strip for usein an instrument system that can produce qualitative CT quantitativeresults. A preferred embodiment of the present invention provides anassay strip including three zones of which two zones are test zones andone of the test zones is a reference zone. A first test zone produces asignal with intensity inversely proportional to analyte concentrationand a second test, zone acts a reference and produces a signal that isdirectly proportional to analyte concentration. The sum of the signalsfrom test zones 1 and 2 is substantially equal at all analyteconcentrations. Quantitative or qualitative results are achieved byinstrumental reading of color intensity on test zone 1, test zone 2 orboth test zones 1 and 2. The results expressed by any one test zone canalso be determined as a proportion of the sum of the actual resultsexpressed by both test zones. Quality reference is achieved byinstrumental reading of both test zones, the sum of which should besubstantially constant within a specified range.

The present invention represents a substantial improvement in the art byproviding methods, assay devices and assay instruments which employ (a)a one-step competitive, lateral flow strip immunoassay, (b) a one-stepinhibition lateral flow immunoassay, (c) a quantitative bibulous stripimmunoassay based on particle detection, (d) two or more detection zonesfor testing, and (e) a reference zone for performing detection tests asa detection zone and perform quality control using the responsescombined from multiple test zones.

The present invention provides an assay which can use specific bindingmembers. A specific binding partner or member, as used herein, is amember of a specific binding pair. That is, two different moleculeswhere one of the molecules through chemical or physical meansspecifically binds to the second molecule. Therefore, in addition toantigen and antibody specific binding pairs of common immunoassays,other specific binding pairs can include biotin and avidin,carbohydrates and lectins, complementary nucleotide sequences, effectorand receptor molecules, cofactors and enzymes, enzyme inhibitors andenzymes, and the like. Furthermore, specific binding pairs can includemembers that are analogs of the original specific binding members, forexample, an analyte-analog. Immunoreactive specific binding membersinclude antigens, antigen fragments, antibodies, and antibody fragments,both monoclonal and polyclonal, and complexes thereof, including thoseformed by recombinant DNA molecules. The term hapten, as used herein,refers to a partial antigen or non-protein binding member which iscapable of binding to an antibody, but which is not capable of elicitingantibody formation unless coupled to a carrier protein.

Analyte, as used herein, is the substance to be detected which may bepresent in the test sample. The analyte can be any substance for whichthere exists a naturally occurring specific binding member (such as, anantibody), or for which a specific, binding member can be prepared.Thus, an analyte is a substance that can bind to one or more specificbinding, members in an assay. Analyte alto includes any antigenicsubstances, haptens, antibodies, macromolecules, and combinationsthereof. As a member of a specific binding pair, the analyte can bedetected by means of naturally occuring specific binding partners(pairs) such as the use of intrinsic factor protein as a member of aspecific binding pair for the determination of vitamin B12, or the useof lectin as a member of a specific binding pair for the determinationof a carbohydrate. The analyte can include a protein, a peptide, anamino acid, a hormone, a steroid, a vitamin, a drug including thoseadministered for therapeutic purposes as well as those, administered forillicit purposes, a bacterium, a virus, and metabolites of or antibodiesto any of the above substances. In particular, such analytes include,but are not intended to ferritin; creatinine kinase MIB (CK-MB);digoxin; phenyloin; phenobarbital; carbamazepine; vancoomyin;gentamicin, theophylline; valproic acid; quinidine; luteinizing hormone(LH); follicole stimulating hormone (FSH); estradiol, progesterone; IgEantibodies; vitamin B2 micro-globulin; glycated hemoglobin (Gly. H10);cortisol; digitoxin; N-acetylprocainamide (NAPA); procainamide;antibodies to rubella, such as rubella-IgG and rubella-IgM; antibodiesto toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosisIgM (Toxo-IM); testosterone; salicylates; acetaminophen; hepatitis Bcore antigen, such as anti-hepatitis B core antigen IgG and IgM(Anti-HBC); human immune deficiency virus 1 and 2 (HIV 1 and 2); humanT-cell leukemia virus 1 and 2 (HTV); hepatitis Be antigen (HBeAg);antibodies to hepatitis Be antigen (Anti-HBe), thyroid stimulatinghormone (TSH), thyroxine (T4), total triiodothyronine (Total T3); freetriiodothyronine (Free T3); carcinoembryoic antigen (CEA); and alphafetal protein (AFP). Drugs of abuse and referenced substances include,but are not intended to be limited to, amphetamine; methamphetamine;barbiturates such as amobarbital, secpbarbital, pentobarbital,phenobarbital, and barbital; benzodiazepines such as librium and valium;cannabinoids such as hashish and marijuana; cocaine; fentanyl; LSD;methaqualone; opiates such as heroin, morphine, codeine, hydromorphone,hydrocodone, methadone, oxycodone, oxymorphone, and opium;phenyloyalidine; and propoxyhene. The details for the preparation ofsuch antibodies and the suitability for use as specific binding membersare well known to those skilled in the art.

The analyte-analog can be any substance which cross-reacts with theanalyte-specific binding member, although it may do so to a greater orlesser extent than does the analyte itself. The analyte-analog caninclude a modified analyte as well as a fragmented, or synthetic portionof the analyte molecule, so long as the analyte-analog has at least oneepitope site in common with the analyte of interest. An example of ananalyte-analog is a synthetic peptide sequence which duplicated at;least one, epitope of the whole-molecule analyte so that theanalyte-analog can bind to an analyte-specific binding member.

The test sample can be derived from any biological source, such as aphysiological fluid, including whole blood or whole blood componentsincluding red blood cells, white blood cells, platelets, serum andplasma; ascites; urine; sweat; milk; synovial raucous; peritoneal fluid;amniotic fluid; percerebrospinal fluid; and other constituents of thebody which may contain the analyte of interest. The test sample can bepre-treated prior to use such as preparing plasma from blood, dilutingviscous fluids, or the like; methods of treatment can involvefiltration, distillation, concentration, inactivation of interferingcompounds, and the addition of reagents. Besides physiological fluids,other liquid samples can be used such as water, food products and thelike for the performance of environmental or food production assays. Inaddition, a solid material suspected of containing the analyte can beused as the test sample. In some instances it may be beneficial tomodify a solid test sample to form a liquid medium or to release theanalyte. The analyte can be any compound, or composition to be detectedor measured and which, has at least one epitope or binding site.

An assay device for the present invention can have many configurations,several of which are dependent upon the material chosen as the porousmember. By “porous” is meant that the material is one through which thetest sample can easily pass and includes both bibulous and non-bibuloussolid phase materials. In the present invention, the porous member caninclude a fiberglass, cellulose, or nylon pad for use in a pour andflow-through assay device having multiple layers for multiple assayreagents; a test strip for wicking or thin layer chromatographiccapillary action (e.g., nitrocellulose) techniques; or other porous oropen pore materials well known to those skilled in the art (e.g.,polyethylene sheet material).

In a preferred embodiment, the present dry reagent assay device uses alateral flow bibulous material with proximal and distal ends, containingat least one central zone along its length. The strip, configuration maybe of any dimensions which provide the desired, number of zones andwhich permit (a) the desired binding reactions to be completed in areproducible manner and (b) detection of the reaction indicator tooccur. Preferably, the present strip is a total of no more than about100 mm in length and about 6 Mt wide, and more preferably, from about 10mm to about 40 mm in length and about 1 mm to about 5 mm wide.

The strip is advantageously integrated into any reflectance basedinstrument, and more preferably, into a disposable electronic assaydevice, such as that described in application Ser. No. 08/111,347,previously incorporated by reference.

The bibulous strip, can comprise a plurality of zones along its length.The zones can contain diffusively or non-diffusively bound reagents.Each zone can, be from about 0.1 mm to about 10 mm wide, more preferablyfrom about 0.25 mm to about 5 mm, wide. There will be a minimum of twozones and a maximum of about 10 or more zones, depending on the numberof assays to be conducted on one bibulous strip.

In a preferred embodiment, the bibulous strip has three zones along itslength. According to this preferred embodiment there are two preferred,configurations including a competitive configuration and an inhibitionconfiguration.

The present invention provides an assay. Method having a, competitivetype configuration. Referring to FIG. 1, at the proximal end 11 of astrip 12 is a first zone 14, comprising a bibulous material containing adiffusively particle-linked antigen. A second zone. 16 is separate anddistinct from the first zone 14, and is located at some distance towardthe distal end 13 of the bibulous strip 12. The second zone 16 includesa bibulous material containing a non-diffusively immobilized antibodycapable of binding the particle-linked antigen and free sample antigen.The bibulous material of the second vine 16 can be the same or differentfrom the bibulous material of the first zone 14.

A third one 18 is separate and distinct from the second zone 16, and islocated at some distance toward the distal end 13 of the bibulous strip12 from the second zone 16. The third zone 18 includes a bibulousmaterial (which may be the same or different from the bibulous materialsof the first and second zones 14 and 16) containing a non-diffusivelyimmobilized first member of a specific binding pair, capable ofspecifically binding to its specific binding partner which is the secondmember of the specific binding pair on the surface of theparticle-linked antigen. This second member of the specific binding pairis not antigenically related to the sample antigen so it will noteffectively compete with the antigen to bind to an anti-antigenmonoclonal antibody.

The sample is applied to the strip 12 at the application site or firstzone 14 which is preferably at the proximal end 11 of the assay strip.The particle-linked antigen is located at or near the application site.The sample containing a sample antigen reconstitutes the driedparticle-antigen conjugate by dissolving or dispersing the conjugate,and the mixture of conjugated and free analyte moves, via bibulouswicking action to the second zone 16, where the free antigen andparticle-conjugated antigen compete for non-diffusively immobilizedantibody at this zone. That portion (e.g., from 0% to 100%) of theparticle-conjugated antigen which binds to the non-diffusivelyimmobilized antibody is retained in the second zone 16. Theantigen-particle conjugate that does not bind to the second zone 16 andmigrates to the third zone la, where substantially all of the portion ofthe particle-conjugated antigen not retained it the tempi zone 16 isbound by the non-diffusively immobilized first member of the specificbinding pair in the third zone 18.

The present invention provides an assay method having an inhibition typeconfiguration. Again referring to FIG. 1, at the proximal end 11 of thestrip 12 is the first zone 14 which includes a bibulous materialcontaining a diffusively immobilized, particle-linked antibody capableof binding sample antigen. The second zone 16 is separate and distinctfrom the first zone 14, and is located some distance toward the distalend 13 of the bibulous strip. The second zone 16 includes a bibulousmaterial containing a non-diffusively immobilized antigen capable ofbeing bound by the particle-linked-antibody. The bibulous material ofthe second one 16 can be the same or different from the bibulousmaterial of the first zone 14.

The third zone 18 is separated and distinct from the second zone 16, andis located some distance toward the distal end 13 of the bibulous strip.The third zone 18 includes a bibulous material which may be the same ordifferent from the bibulous materials of the first and second zones 14and 16 containing a, non-diffusively immobilized first member of aspecific binding pair capable of specifically binding to its specificbinding partner which is the second member of the specific binding pairon the surface of the particle linked antigen. This, second member ofthe specific binding, pair is not antigenically related to the sampleantigen so it will not effectively compete with the antigen to bind toan anti-antigen monoclonal antibody.

The fluid sample is applied to the strip at the application site ispreferably in the proximal end of the assay strip. The application siteis where the particle-linked antibody is located. Sample antigen whichmay be present in the sample reconstitutes the particle-antibodyconjugate and is bound by the conjugate. The boundantigen:antibody-particle complex, as well as unbound antibody-particlecomplex, are transported or migrate via capillary or wicking action tothe second zone 16, where substantially all of the freeantibody-particle conjugate is bound by the non-diffusively immobilizedantigen. The bound sample antigen:antibody-particle complex migratesthrough the second zone 16 to the third zone 18, where substantially allof it is bound by the non-diffusively immobilized first member of thespecific binding pair.

In the preferred embodiments described above, the amount of a detectableresponse or signal present at the second zone 16 is an inverse measureof the sample analyte concentration, and the amount of the detectableresponse or signal at the third zone 18 is a direct measure of thesample analyte concentration. The detectable responses or signalscombined from second and third zones 16 and 18 are approximatelyconstant across the entire range of sample analyte concentration. Thistotal detectable response or signal serves as a reference mechanism forboth the assay procedure and reagent quality. Thus, if the total signalis below a specified range, the user is notified of an error.Furthermore, the specific reason for the incorrect assay procedure canbe identified. For example, the error can be identified as operationoutside the specified temperature and/or humidity range, insufficientsample volume, expired reagents, or the like.

The assay quantitation can be determined by reading the second zone 16,the third zone 18, or both second or third zones 16 and 18. The sampleconcentration output is a result of a calibration algorithm related tothe second zone 16 alone, the third zone 18 alone or both second andthird zones 16 and 18. This can result in a more reliable quantitativeanalyte, concentration result. The summation of the detectable responsesor signal from second and third zones 16 and 18 to produce asubstantially constant total signal regardless of analyte concentrationprovides a reference mechanism for accurate assay performance.

The above strip configurations are advantageously used in the integratedassay instrument described in U.S. application Ser. No. 08/111,347.Although the chemistry and configurations of the present invention maybe used in an integrated assay device, the present invention can be usedin any other instrumented reflectance or transmission meter as areplaceable reagent. Thus, the present invention also encompassesintegrated assay instruments and analytical assay instruments comprisingthe present assay device.

The present invention preferably uses particle detection for adetectable response or signal in each test zone related to the level ofanalyte in the sample. Other means for providing a detectable responsein the test zones are suitable for use in the present invention. Forexample, and not for limitation, the analyte may be labelled with anindicator to measure electrical conductance or the reflectance orabsorption of a characteristic light wavelength. As use herein,“indicator” is meant to include all compounds capable of labelling theanalyte or conjugate thereof and generating a detectable response orsignal indicative of the level of analyte in the sample.

The present assay device and method represents a substantial improvementin the reliability of single-use diagnostic devices utilizingchromatographic strip; binding assays for determining the presence oramount of an analyte in a sample, e.g. taken from a medical patient.

The assay devices include a bibulous substrate to which members ofspecific binding pairs, which may be labeled, are diffusively ornon-diffusively immobilized. Non-diffusive immobilization can beconducted by adsorbing, absorbing, crosslinking or covalently attachinga reagent such as a labeled member of a binding pair to the bibuloussubstrate.

Diffusive immobilization can be conducted by formulating one or moreassay reagents to be immobilized. Examples of formulating the reagentsinclude dissolving in a suitable solvent such as water, a C₁-C₄ alcoholor mixture thereof, along with any desired additives. The resultingformulation is applied to the bibulous material of the assay device inone or more desired locations, and then, the bibulous material is dried.Diffusive immobilization allows rapid reconstitution and movement ofreagents, whether reacted or unreacted, through the bibulous substrate.

The present invention also includes to a one-step lateral flow assaystrip comprising two or more test zones for each analyte and a particledetection system that is quantitatively read by a reflectance typeinstrument. FIGS. 1-7 show various embodiments of strip configurationssuitable for immunoassay devices and methods.

The present immunoassay configurations can measure a wide variety ofanalytes. The immunoassays can be set up to be either qualitative (e.g.,as in the cases of HCG (pregnancy) assays, assays for knownmetalabolites associated with drugs of abuse or for known antigensassociated with infectious diseases) or quantitative (e.g., in the caseof bone collagen N-telopeptide (NTx; assayed as a marker for boneresorption), theophylline, digoxin, quantitative HCG (ectopicpregnancy), C-reactive protein, CKMB and Troponin).

The present device May be used on-site in the home and in physician'soffice, or in remote locations in emergency medicine. Therefore, thedevice may advantageously include, sample pre-treatment as previouslydefined, as well as a sample withdrawal device (e.g., a fingerstick) orany combination thereof, sample pretreatment can also adjust the pH towithin a specified range, reference salt concentration, turbidity and/orviscosity, and/or reduce or remove immunochemical cross-reactants. Eachimmunoassay configuration shown in FIGS. 1-7 can include samplepretreatment, including one or more chemical, filtration or separationmeans, or any combination thereof.

The present invention provides a device which can be used to determinethe presence of multiple analytes in a test sample. One test zonecorresponds to each analyte selected, for determining its presence. Eachtest zone receives and contacts the sample and a labeled indicatorreagent corresponding to the selected analyte with a test zone reagentcorresponding to the selected analyte. The test zone reagent correspondsto the selected analyte reacting; in the presence of the sample and thelabeled, indicator reagent corresponding to the selected analyte to forma corresponding test zone reaction product and a corresponding test zonedetectable response inversely related to the selected analyte level inthe sample.

One reference zone receives the labeled indicator reagent not reactedwith its corresponding-test zone reagent from all the test zones. Thereference zone contacts each labeled indicator reagent with acorresponding reference zone reagent. Each reference zone reagent reactsin the presence of the corresponding labeled indicator, reagent to forma corresponding reference zone reaction product and a correspondingreference zone detectable response related to each selected analytelevel in the sample and proportionately related to the correspondingtest zone detectable response to establish a substantially constanttotal detectable response, for a pre-determined range of each selectedanalyte. The detectable responses from each test zone are separatelycombined with the detectable result from the reference zone to determineeach selected analyte level in the sample.

Referring again to FIG. 1, a top surface view of an embodiment isillustrated having a typical structure with three zones including twotest zones for a single analyte, and FIG. 2 shows a top surface view ofa similar device with four zones including three test zones for twoanalytes. The same reference numerals are used to identify the sameelements between the Figures.

In FIG. 1, the first zone 14 of strip 12 is located at or slightlydownstream from the sample application, site at the proximal end 11 ofthe strip, and second zone 16, located downstream from the first zone14, may be either directly adjacent to or separated by a bibulous spacerbut in fluid communication with the first zone 14. The third zone 18,located downstream from zones 14 and 16, may be either directly adjacentto the second zone 16 or separated but in fluid communication with thesecond zone 16. The third zone 18 acts as a reference zone for thesecond zone 16. As used herein, “fluid communication” refers to a director indirect contact of bibulous material which permits a, fluid sampleto flow from the sample application site or first zone 14 of the device;through the various zones of the device, to the periphery of the device.

FIG. 2 is a similar construction with an additional fourth tone 20,located downstream from zones 14, 16 and 18, which may be eitherdirectly adjacent td the third zone 18 or separated but in fluidcommunication with the third zone 18. The fourth zone 20 acts as areference zone for each of the second and third zones 16 and 18. Allzones are in fluid communication, both with each other and with thesample application site.

The sample application site is preferably in the first zone 14, oralternatively, can be a separate area directly adjacent to and upstreamfrom the first zone 14 (preferably still positioned at the proximate endof the strip). Zones 14, 16, 18, and 20 may be of any dimensions whichprovide adequate detection of the indicator in the assay(s), andpreferably are from about 0.05 cm to about 1.5 cm in length (morepreferably about 0.1 cm to about 1.0 cm in length).

The overall dimensions of the strip may be any dimensions which provideadequate spacing and resolution for conducting the assay(s). Preferably,however, the length of the strip is in the range of about 2 cm to about10 cm (more preferably about 2 cm to about 4 cm) and the width can beabout 0.1 cm to about 1.5 cm (more preferably about 0.2 cm to about 0.5cm). The strips shown in FIG. 1 and FIG. 2 are, for example, about 3 cmlong and about 0.3 cm wide.

The strip can be one continuous section of bibulous material or becomposed of one, two, three or more sections. Each zone may be aseparate bibulous material where each zone is in fluid communicationwith adjacent zones, or two or more adjacent zones may share a commonmaterial, with the other zones being different materials.

The assay strip including each of the zones can be composed of the tameor different bibulous materials. The bibulous material permits fluidcommunication, between the various zones, spacers (if present) andsample application site by wicking or capillary action upon-applicationof a fluid sample. Examples of materials that tan be used include butare not limited to: cellulose papers such as WHATMAN 1C, 2C, 4C, 31ET,S&S 903C, GB002; membranes such as S&S nitrocellulose, celluloseacetate, regenerated cellulose at pore sizes from 1 μm to 20 μm, Pallnylon at, pore, sizes of 1μ to 20μ including BIODYNE® A, B, C orIMMUNODYNE® ABC, Gelman ULTRABIND®, Millipore IMMOBILON®; compositepapers or membranes made from mixtures of glass fiber, plastic or metalfiber or synthetic or natural mesh or fabric made from cotton,cellulose, polyethylene, polyester or nylon.

Zones 14, 16, 18 and 20 of FIGS. 1 and 2 can contain, reagentsdiffusively or non-diffusively bound including, but not limited to,antibodies, antigenS, enzymes, substrates, small molecules, proteins,recombinant proteins, viral, or bacterial lysate, receptors, sugars,carbohydrates, polymers like PITA and detergents.

FIGS. 3-5 shows exploded lengthwise cross sections of the embodiment ofFIG. 1. The backing 24 in FIGS. 3-5, may provide structural support forthe bibulous material. Backing 24 can be of any convenient materialthat, provides support for the assay matrix and is preferably a plastic,such as cellulose acetate, polyester, vinyl or the like, or a syntheticor natural fabric or mesh. Backing 24 has a thickness sufficient tosupport the assay material, and preferably has a thickness of from about0.002 inch to about 0.015 inch (more preferably about 0.005 inch toabout 0.010 inch thick). However, if the bibulous material is itselfsufficiently rigid, or is supported by other mechanical means, then abacking is not necessary.

An adhesive 22 can, be interposed between backing 24 and the bibulousmaterial, to promote, adhesion of these layers. Adhesive 22 can be anydouble stick adhesive, such as 3M 415, 443, 9460 or the like.Alternatively, a membrane that is cast during manufacturing to a plasticsupport such as S&S PB-NC can be used. In this case, an adhesive is notnecessary.

FIG. 3 shows an exploded lengthwise cross section of the embodiment ofFIG. 1 which does not have sample pretreatment. In this configuration,the sample is introduced at the proximal end 11 of the strip in the areaof the first zone 14.

FIG. 4 shows an exploded lengthwise cross section of the embodiment ofFIG. 1 with one type of sample pre-treatment. The sample pre-treatmentcan include any combination of chemical, filtration or separationtreatments, including blood separation. The sample treatment zone may becomposed of one, two, or several layers of depth filter material 26(such as glass fiber, metal fiber, synthetic fiber, paper, or natural orsynthetic fabric) and a membrane 28 (such as S&S cellulose, acetate,nitrocellulose, regenerated cellulose having an average pore size offrom about 0.2 μm to about 7 μm, and Nucleopore or Poreticspolycarbonate at pore sizes of about 0.2 μm to about 5 μm).

The layers of materials 26 and 28 can contain any number of assayreagents including but not limited to buffers, salts, proteins, enzymesand/or antibodies (either or both of which may be diffusively ornon-diffusively bound to a particle or the bibulous material), polymers,small molecules, or any combination thereof. If red blood cells are tobe separated, then layers of materials 26 and 28 function to removesubstantially all of the red blood cells from the blood-sample, leavingplasma to operate in the assay.

As Shown in FIG. 4, sample filtration 26 and 28 is positionedimmediately above and in fluid communication with the first zone 14. Thesample filtration 26, 28 can be of any dimensions which effectivelyremove red blood cells from a whole blood sample to be assayed, and arepreferably from about 0.2 to about 1 cm in length. The sample filtrationcan be secured with adhesive or be held in place by the instrumenthousing. The adhesive for affixing the sample filtration means in placemay be any adhesive, such as epoxy, hot melt glue, or the like, or anadhesive tape such as that made by the 3M company.

FIG. 5 shows an exploded lengthwise dross section of the embodiment ofFIG. 1 with a second type of sample pre-treatment and transport means.The sample treatment in the device of FIG. 5 can include any combinationof chemical, filtration or separation means, including blood separationmeans. The sample treatment and transport device, of FIG. 5 includes asample application zone at filter 32, a membrane 34, a transport mesh36, a second filter 38 and a membrane 40.

The filter 32 can be composed of one, two, three or more layers of anybibulous material, preferably a depth filter such as glass fiber, metalfiber, synthetic fiber, paper, or natural or synthetic fabric. Filter 34can be one or several layers and is composed of any microporous membranesuch as S&S cellulose acetate, nitrocellulose, regenerated cellulose, atpore sizes from about 0.2 μm to about 7 μm, Nucleopore or Poreticspolycarbonate at pore sizes of about 0.2 μm to about 7 μm.

Although filters 32 and 34 are shown in FIG. 5, one or both of theselayers may not be necessary and can be excluded. In the case where bothfilter layer 32 an 34 are excluded, the sample will be applied directlyto the transport layer 36.

The sample transport layer 36 is designed to accept the sample, eitherdirectly or through the filter layers 32 and 34, and move ithorizontally to the area of filter 38. This sample movement may takefrom about 2 seconds to about 10 minutes, preferably from about 2seconds to about 5 minutes, and more preferably from about 5 seconds toabout 2 minutes.

The sample transport is composed of any bibulous material including, butnot limited to, fabric or mesh that is woven or cast, synthetic ornatural, and made of cotton, nylon, polyester, polypropylene,polyethylene or the like; paper such as Whatman 31ET or 3; glass fibersuch as Whatman GFA, GFD, S&S 3362 or 32; plastic fiber, metal fiberand/or any synthetic membrane. The sample transport area can beuntreated, or may have diffusively or non-diffusively immobilizedtherein one or more reagents such as stabilizing proteins, detergents,anticoagulants like heparin or EDTA, precipitating reagents, salts,proteins, enzymes, antibodies, enzyme-particle conjugates,antibody-particle conjugates, antigen-particle conjugates, red cellagglutinating agents like wheat germ lectin or anti-human RBC, polymersand/or small molecules.

The sample transport layer/zone 36 has dimensions sufficient to permitany desired sample pre-treatment without adversely affecting assayreactions and indicator measurements, but is preferably about 0.5 cm toabout 5 cm (more preferably about 1 cm in length) in length and about0.1 to about 1.5 cm (more preferably about 0.2 to about 0.5 cm) inwidth.

The filter 38 can be composed of one, two, three or more layers of anybibulous material, preferably a depth filter such as glass fiber, metalfiber, synthetic fiber, paper, or natural or synthetic fabric. Filter 40can be one or several layers and is composed of any microporous membranesuch as S&S cellulose acetate, nitrocellulose, regenerated-cellulose atpore sizes from about 0.2 μm to about 7 μm, Nucleopore or Poreticspolycarbonate at pore sizes of about 0.2 μm to about 5 μm. Althoughfilters 38 and 40 are shown in FIG. 5, one or both of these layers maynot be necessary and can be excluded. In the case where both filterlayers 38 and 40 are excluded then the sample will be transporteddirectly from the transport layer 36 to the first zone 14. All of thesample treatment and transport materials 32, 34, 36, 38, and 40 are influid communication with each other and with the first zone 14.

If the device is used for blood separation then it will function toremove substantially all of the red cells from the blood sample, leavingplasma to operate in the assay. The red cells can be substantiallyremoved by filters 32 and 34 prior to the sample contacting thetransport mesh or the red cells can be removed by filters 38 and 40 inwhich case the whale blood will travel on the transport layer. In apreferred embodiment, filters 32 and 34 are absent and sample blood orurine or any other body fluid is applied directly to the transport layerand sample treatment, filtration and/or blood separation occurs atfilters 38 and 40.

Filters 32, 34, 38, and 40 have dimensions sufficient to permit anydesired sample pre-treatment without adversely affecting assay reactionsand indicator-measurements, but preferably are about 0.2 CM to about 2cm (more preferably about 0.25 to about 0.75 cm) in length and about 0.1to about 1.5. cm (more preferably about 0-2 to about 0.5-cm), in width.The components of the sample treatment means and transport means of FIG.5 can be secured with adhesive or held in place by a rigid housing. Theadhesive can be any convenient adhesive including epoxy, hot melt glue,or the like, or may be an adhesive tape such as those, made by 3Mcompany.

Referring now to FIGS. 6 and 7, each of these immunoassay formats canhave a sample treatment means and/or a transport means as described forthe assay devices in FIGS. 4 and 5. Alternatively, they may have asample treatment means as described for the assay device in FIG. 3.

FIGS. 6 and 7 show two embodiments of a quantitative assay to measurethe concentration of the crosslinked bone collagen telopeptide (NTx) inurine, whole blood, plasma or serum. NT is a product of bone resorptionand is known to be present in urine and blood. The concentration of NTxis a direct measure of the rate of bone resorption and is a usefulmarker for (a) the onset of osteoporosis and (b) monitoring the progressof therapy for osteoporosis. Although NTx is shown as an example assayaccording to the present invention, it is understood that any analytecan be quantitatively or qualitatively measured.

The assay strips of FIGS. 6 and 7 each have two test zones. The two-testzone design provides improved performance in quantitative assays andimproved reliability, since the sum of the signals from both test zonesis substantially constant regardless of the analyte/antigenconcentration, thus providing a robust quality reference and assuringaccurate assay operation.

FIG. 6 is a top surface view of an inhibition type immunoassayconfiguration, a preferred embodiment of the present invention. In theassay strip 50, zone 52 contains a diffusively bound anti-NTx antibody(of any other antibody), conjugated to colloidal gold, colored latexbeads or an enzyme. The diffusively immobilized anti-NTx-particleconjugate can also be located on filters 26 or 28 of the device of FIG.4 or on filters 32, 34, 38 or 40 or transport layer 36 of the device ofFIG. 5.

The antibody can be monoclonal (e.g., derived from fusion of spleencells from an immunized mouse with a suitable immortal cell line inaccordance with known methods; see Kohlstein and Milner, 1975) orpolyclonal prepared from any suitably immunized animal species inaccordance with known methods).

A preferred embodiment uses conjugates of anti-NTx antibody to particlesof colloidal gold, or to blue or black latex beads. Particles can befrom about 5 nm to about 2000 nM in diameter (Mote preferably from about5 nm to about 500 nm in diameter).

Diffusive immobilization can be conducted by formulating the assayreagent(s) to be immobilized (e.g., by dissolving in a suitable solvent,such as water, a C₁-C₄ alcohol or mixture thereof, along with anydesired, additives), applying the resulting formulation to the bibulousmaterial of the membrane, filter or transport layer in the desiredlocation(s), and drying the material. Suitable additives may include,detergents, proteins, blocking agents, polymers, sugars or the like.Alternatively the additive(s) and assay reagent(s); may be applied tothe membrane, filter or transport layer by precoating with a “blockingagent”, water soluble polymer, sugar or detergent, followed bydepositing the conjugate or conjugate formulation and drying thematerial.

Zone 54 is the first test zone of strip 50. Zone 54 containsnon-diffusively bound NTX, NTx-macromolecule conjugate or NTx-particleconjugate. NTx is conjugated to a macromolecule or particle to help inthe immobilization of the NTx peptide to the membrane (bibulousmaterial) surface. Suitable macromolecules which can be used for NTxconjugation include any large molecule capable of adsorption or covalentbinding to the membrane, including but not limited to: bovine serumalbumin (BSA), keyhole limpet hemocyanin (KLH), immunoglobulin G (IgG),mouse IgG, bovine gamma globulin (BGG), lactalbumin, polylysine,polyglutamate, polyethylenimine, or aminodextran. Suitable particleswhich can be used for NTx conjugation can include particles of about1-20 μm in diameter, including, but not 0.39 limited to, latexparticles, microcapsules, liposomes or metal sol particles.

Non-diffusive immobilization can be accomplished by covalentlyattaching, adsorbing or absorbing the NTX, NTX-protein conjugate or NTXparticle conjugate to the membrane. Suitable membranes for adsorption orabsorption include, but are not limited to, S&S nitrocellulose andcellulose acetate at pore sizes from 0.45 μm to. 12 μm, and Pall nylonat pore sizes of 0.45 μm to 20 μm (such as BIODYNE A, B, and C).Suitable membranes for covalent attachment include, but are not limitedto, membranes such as Millipore IMMOBILON®, Gelman ULTRABIND® and PallIMMUNODYNE® ABC. Alternatively, the antigen, antigen-protein conjugateor antigen-particle conjugate can be covalently attached to the membraneby chemically activating the membrane or paper prior to applying asolution or formulation of. antigen/conjugate. Covalent attachment ofthe NTx peptide to the membrane occurs through a linkage to the primaryamine on the NTX molecule.

Zone 56 is the second test zone of strip 50. Zone 56 contains anon-diffusively bound member of a specific binding pair capable ofbinding to a complementary member of the specific binding pair which isnot related, to the sample analyte/antigen on the surface of theparticle-linked antibody.

For example, if the particle-linked antibody is a mouse monoclonalantibody, then the non-diffusively complementary binding partner in zone56 can be any anti-mouse polyclonal or monoclonal antibody, includingbut not limited to: goat-anti-mouse, sheep-anti-mouse, cow anti-mouse,rabbit-anti-mouse, monoclonal rat anti-mouse or any other anti-mousespecies antibody.

Alternatively, a generic binding partner such as Protein A, Protein G orProtein A/G (e.g., obtained from Pierce) can be non-diffusivelyimmobilized at zone 56, as long as it binds the particle-antibodyconjugate. Lectins can also be immobilized at zone 56, provided that theparticle-antibody conjugate can be bound at this zone. Biotin, avidin orstreptavidin can be linked to particle or to the particle-linkedantibody, and the complementary binding partner may then benon-diffusively immobilized at zone 56.

For example, if biotin is conjugated to the particle along, with theantibody, thus producing an anti-NTx-particle-biotin conjugate, thenavidin or streptavidin can be non-diffusively immobilized, at zone 56and used to capture particles not bound in zone 54. Any non-humanantigen, including proteins or small molecules such as dinitrophenol,known dinitrophenyl group-containing molecules or fluorescein can beco-conjugated with anti-NTx to the particle. The complementary antibodycan then be immobilized to zone. 56, the requirement being that theparticle conjugate hot bound in zone 54 is substantially all captured(bound) in zone 56 in the assay.

In the assay operation of FIG. 6, the sample is introduced to theproximal end of the assay strip in the area of the particle-linkedantibody conjugate zone 52. The sample can be applied directly, or canbe pre-treated, filtered, and/or separated as described above. The fluidsample (sample antigen, in this case NTx) then reconstitutes theparticle-antibody (particle-anti-NTX) conjugate, and any antigen in thefluid sample is bound by the conjugate in zone 52. The particle-antibodyconjugate is applied in excess, such that most of the antigen is boundby the conjugate.

The bound antigen:antibody-particle complex (NTx:anti-NTX-particle), aswell as unbound antibody-particle (anti-NTx-particle) conjugate, migratefrom zone 52 via capillary action to zone 54, where substantially all ofthe free antibody-particle conjugate is bound by the non-diffusivelyimmobilized antigen (NTx) at this site. The antigen:antibody-particlecomplex cannot bind to the non-diffusively immobilized antigen at zone54 since the binding sites are occupied by sample antigen. Consequently,the antigen antibody-particle complex migrates via capillary action tozone 56 and is substantially all bound by the non-diffusivelyimmobilized complementary member of the specific binding pairimmobilized at this site.

At zero sample antigen concentration, the binding sites on theparticle-antibody conjugate are free, and the particles are mostly boundat zone 54, where a dark color is produced. At very high sample antigenconcentrations, the binding sites on the particle-linked antibody aremostly occupied, and the particles move past zone 54 and aresubstantially all bound at zone 56. Intermediate concentrations ofsample antigen result in a predictable response relative to the boundparticle signals at zones 54 and 56. In general, low sampleconcentrations result in high signal in one 54 and low signal in zone56. As analyte/antigen concentration increases, the signal in zone 54becomes progressively lower, and the signal in zone 56 becomes.correspondingly higher. The total signal, which is the sum of signalfrom zones 54 and 56, remains substantially constant across the entireconcentration range. This provides a reliable quality reference for theassay result, since the sum of the signals must stay within a specifiedrange. Otherwise, an assay failure is indicated.

Assay calibration and sample quantitative measurement can be, achievedusing zone 54 alone, zone 56 alone, or both zones 54 and 56. Undercertain conditions, one test zone may produce better performance in aparticular analyte/antigen concentration range, and the other test zonemay produce better performance in a different analyte/antigenconcentration range. In this case, a hybrid calibration can be done thatuses the optimal calibration range of each zone. Thus, the presenttwo-test zone measurement provides substantial improvements overpreviously described methods.

FIG. 7 is a top surface view of a competitive-type immunoassayconfiguration, another preferred embodiment of the present invention. Inthe assay strip 60, zone 62 contains diffusively bound NTx (or othersample antigen) conjugated to colloidal gold, colored latex beads or anenzyme. The NTx can be coupled directly to the particle. Alternatively,NTx can be coupled indirectly to the particle through the macromoleculemoiety of a macromolecule-NTX conjugate. The macromolecule used for NTxconjugation can be any large molecule capable of adsorption or covalentbinding to the particle, including but not limited, to: bovine serumalbumin (BSA), keyhole limpet hemocyanin (KLH), immunoglobulin G (IgG),mouse. IgG, bovine gamma globulin (BGG), lactalbumin, polylysine,polyglutamate, polyethylenimine, or aminodextran.

A preferred embodiment uses conjugates of NTx to particles of colloidalgold, or to, blue or black latex beads. Particles can be from about 5 nmto about 2000 nm in diameter (more preferably from about 5 nm to about500 nm in diameter).

The NTx-particle conjugate can also be diffusively immobilized onfilters 26 or 28 of the device of FIG. 4 or on filters 32, 34, 38 or 40or transport layer 36 of the device of FIG. 5. Diffusive immobilizationcan be accomplished as described above.

Zone 64 is the first test zone of strip 60. Zone 64 containsnon-diffusively bound anti-NTx. Non-diffusive immobilization can beaccomplished by covalent attachment or adsorption of the anti-MIX to themembrane as described, above. Alternatively, anti-NTx can be conjugatedto another protein, and this conjugate is then adsorbed to the membrane.Adsorption can be accomplished using membranes including, but notlimited to, S&S nitrocellulose and cellulose acetate at pore sites from0.45 μm to 12 μm, and Pall nylon at pore sizes of 0.4.5 μm to 20 μm(such as BIODYNE A, B, and C). Covalent attachment can be accomplishedusing membranes such as Millipore IMMOBILON®, Gelman ULTRABIND® or PallIMMUNODYNE® ABC, or by chemically activating the membrane or paper priorto contacting the antibody with the membrane or paper.

Zone 66 is the second test one of strip 60. Zone 66 contains anon-diffusively bound member of a specific binding pair such as anantibody or an antigen which is not immunologically related to thesample analyte/antigen, avidin, biotin, Protein A or G, lectin or thelike) which binds to a complementary member of the specific binding pairon the surface of the particle-linked antigen. For example, if theparticle is linked to both antigen and protein (e.g., anantigen-macromolecule-particle conjugate), then an antibody to thatprotein can be non-diffusively immobilized in zone 66.

Furthermore, for example, if NTx is conjugated to mouse IgG, and theparticle is linked to this, conjugate (NTx-mouse IgG-particle), then anyanti-mouse antibody can be non-diffusively immobilized at zone 66. Anyprotein carrier can be used to conjugate to NTx, and the correspondingantibody (to the protein carrier) it then non-diffusively immobilized tozone 66.

Alternatively, any generic binding partner such as Protein A, Protein Gor Protein A/C (e.g., obtained from Pierce) can be non-diffusivelyimmobilized at zone 66 as long as it binds the particle-antigenconjugate. Lectins can also be immobilized at zone 66, provided that theparticle-antigen conjugate can be bound at this zone.

Biotin, avidin or streptavidin can be conjugated to the particle-linkedantigen, and the complementary binding partner can then benon-diffusively immobilized in zone 66. For example, if biotin isconjugated to the particle along with the antigen (in this case NTx),thus producing a biotin-particle-NTx conjugate, then avidin orstreptavidin can be non-diffusively immobilized in zone 66.

Any non-human antigen, including proteins or small molecules, such asdinitrophenoi, known dinitrophenyl group-containing molecules orfluorescein, can be co-conjugated with NTx to the particle, and thecomplementary antibody can be immobilized in zone 66, the requirementbeing that the particle conjugate, that is not bound in zone 64 issubstantially all captured (bound) in zone 66 in the assay.

in the assay operation, the sample is introduced to the proximal end ofthe assay strip in the area of the particle-linked antigen conjugatezone 62. The sample can be directly applied, or alternatively, it can bepre-treated, filtered, and/or separated as described above. The fluidsample (which may contain antigen, in this case NTx) then reconstitutesthe particle-antigen (particle-protein-NTx) conjugate, and the mixtureof particle-protein-NTx and free analyte (NTx) moves via capillarymigration or bibulous wicking action from zone 62 to zone 64, where thefree antigen and particle-conjugated antigen compete for bon-diffusivelyimmobilized antibody. The antigen-particle conjugate that does not bindto one 64 migrates to zone 66 and is substantially all bound by thenon-diffusively immobilized member of the specific binding pairimmobilized at this site.

At zero sample analyte/antigen concentration, the particle-antigenconjugate is mostly bound in zone 64, resulting in a dark color beingproduced in this zone. At very high sample analyte/antigenconcentrations, the analyte/antigen occupies most of the binding sitesof zone 64, causing the particle-linked conjugate to move past zone 64to zone 66, where it is substantially all bound. Intermediateconcentrations of sample analyte/antigen result in a predictableresponse relative to the bound particle signals. in zones 64 and 66.

In general, low analyte/antigen concentrations result high signal inzone 64 and low, signal in zone 66. As sample analyte/antigenconcentration increases, the signal in zone 64 becomes progressivelysmaller, and the signal in zone 66 becomes. correspondingly higher. Thetotal signal or detectable response (i.e., the sum of the signals fromzones 64 and 66), remains substantially constant regardless of theanalyte/antigen concentration (e.g., across the entire concentrationrange of from 0 to about 100 mM). This provides a reliable assay resultand quality, reference since the sum must stay within a specified range,otherwise an assay failure is indicated.

Assay calibration and sample quantitative measurement can be achievedusing zone 64, alone, zone 66 alone- or both zones 64 and 66. Undercertain conditions, one test zone may produce better performance in aparticular analyte/antigen concentration range, and another test zonemay produce better performance in a different analyte/antigenconcentration range. In this case, a hybrid calibration can be done thatuses the optimal calibration range of each zone. Thus, the presenttwo-test zone measurement provides substantial improvements overpreviously described methods.

The present test strip, may be advantageously used in an instrumentwhich reads the signals in zones 64 and 66. Thus, the indicator signalsneed not be visually detectable.

Having generally described the present invention, a furtherunderstanding can be obtained by reference to the following specificexamples, which are provided herein for purposes of illustration onlyand are not intended to be limiting of the present invention. Unlessotherwise specified, temperatures are in degrees Centigrade and percentsare weight percents.

Example 1

The devices of the embodiments shown in FIGS. 1-7 are quantitative orqualitative immunoassay strips. The assay strips shown in theseembodiments can be configured by one of two assembly methods.

In a first strip assembly method, the strip is composed of severalSeparate bibulous membrane sections in fluid communication by laminationto a plastic strip. The reagents can be diffusively or non-diffusivelyimmobilized to the membrane prior to lamination. Alternatively, thereagents can be immobilized after lamination.

For convenience, the assay strips are constructed in bulk in a card formwith the discrete assay zones forming lines along the length of thecard. Each card can be of any convenient size, depending only on thelength of the assay strip and the number of assay strips desired. Forexample, if the assay strip (as shown in FIG. 1) is 6 cm long and 0.5 cmwide, then the card can be 6 cm by 10 cm (20×0.5 cm), thus providing 20strips. Two sizes of strips were used in the examples below. For a stripsize of 6 cm long by 0.5 cm wide, the card was 6 cm by 10 cm (yielding20 strips); for a strip size of 3 cm long by 0.3 cm wide, the cards were13 cm by 6 cm (allowing 20 strips).

In a second strip assembly method, the strip is one continuous materialthat may optionally be laminated or cast to a plastic support. Thereagents are diffusively or non-diffusively immobilized to a continuousassay strip, and are applied to the strip using a process that “prints”the reagents in discrete zones along the length of the strip. The assaystrips may be constructed in bulk in a card form in accordance with thefirst strip assembly method described above.

The following strip assembly and reagent immobilization methods wereused in the construction of the present invention.

One method of constructing a strip assembly for the present inventionlaminated the membrane or paper to a plastic backing by joining themembrane or paper assay matrix to a sheet of polyvinyl acetate (0.01″thick) using a double-stick adhesive or a transfer adhesive. This isillustrated in FIGS. 3-5.

A card of polyvinyl acetate sheet (0.01″ thick) was cut to about 6 cm by10 cm (or 3 cm by 6 cm). The size of the card varied, depending on thedesired assay card site. The polyvinyl acetate backing was marked withpencil lines along the length at appropriate positions indicating thelocation of the various assay strip zones. Double stick adhesive, suchas 3M 415 to the polyvinyl acetate, was applied so as to, cover thesurface with the pencil lines, and firm pressure was applied with aroller assembly making sure to eliminate the formation of bubbles. Therelease liner of the double-stick adhesive was removed and the membraneor paper assay sections applied to the correct location, guided by thepencil lines, and firm pressure was applied with, a. roller assemblymaking sure to eliminate bubbles. Care was taken to ensure that eachsection of the bibulous assay matrix was in fluid communication with itsneighbor. Finally, individual assay strips were cut, each 0.5 cm wide(or 0.3 cm) along the length of the card, resulting in assay strips 0.6cm long by 0.5 cm wide (or 3 cm long by 0.3 cm wide). This wasaccomplished using a die cutter or a standard paper cutter.

A non-diffusive immobilization was accomplished using a variety ofmethods. In a preferred method, nitrocellulose or nylon membrane (poresizes of 0.45 μm to 12 μm) was incubated with a protein, protein-haptenconjugate, peptide; small molecule or the like (immobilization compound)to be non-diffusively immobilized, in 50 mM sodium-phosphate, pH 7, for60 minutes. The membranes were then washed twice in 50 mM. sodiumphosphate pH 7, 0.1 M NaCl (PBS) for 15 minutes and preserved in 5 Mg/mLBSA, 1% sucrose solution for 10 minutes. Drying was done at 50° C. for15 minutes or until dry.

In a second non-diffusive immobilization method, an applicator (e.g., a.fountain pen, a pad printer, pipette, air brush, inkjet print head orthe like) was used to accurately measure the reagents onto appropriatezones of the as matrix. In this case, the immobilization compound wasdiluted to between 0.01 mg/mL and 10 mg/mL with 50 mM phosphate, pH 7,and introduced into the application device. The application device wasthen positioned. above the appropriate assay zone and the immobilizationmaterial was coated onto the assay matrix. The strips were washed usingPBS and preserved with BSA/sucrose prior to drying, or they may not bewashed or preserved. The assay membrane was then-dried at 50° C. for 15minutes or until dry. This method provides flexibility in “printing”reagents in a referenceled manner at any location along the assay strip.

In a third method, the immobilization compound can be covalently coupledto latex microparticles of about 1-20 μm and these part idles are drawninto the membrane matrix using suction or pressure. The microparticlemethod is accomplished by first covalently immobilizing the desiredprotein to microspheres with carboxyl functional groups as follows: To asuspension of 10 μm microspheres-COOH (e.g., Bangs. Laboratories stock#PO100000CN) add 1.1 molar equivalents (relative to the COOH groups onthe bead surface) of 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDAC,Sigma E 0388) and 1.1 molar equivalents of N-hydroxysuccinimide (NHS,Pierce 24500) in 0.1 M sodium phosphate, pH 7.0, at room temperaturewith stirring for 30 minutes. Add this mixture to a stirring solution ofthe desired protein in 0.1 M sodium phosphate, pH 7.0, (the protein isat a 10 fold molar excess over the COOH functional groups on the beadsurface). Allow to react for 2 hours at room temperature and then purifyby centrifuging, followed by washing and dialysis. The microparticlesnow have the desired protein covalently immobilized. Theprotein-particle suspension is then mixed and 2-10 μL is picked up usinga pipette. The membrane or paper assay strip is placed on a sinteredglass filtration platform with vacuum and the bead-protein suspension isapplied from the pipette across the assay strip in the correct location.The vacuum pressure draws the conjugated beads into the matrix of themembrane or paper where they are mechanically non-diffusivelyimmobilized. Alternately these beads can be applied to the membraneusing an air brush or inkjet type print head.

A fourth type of non-diffusive immobilization, involved covalentattachment of the immobilization compound to the assay matrix. This wasaccomplished by contacting a solution or formulation of the compound(s).to be immobilized with a commercially available activated membrane orpaper, such as Pall IMMUNODYNE® ABC, Gelman ULTRABIND® or MilliporeIMMOBILON®, using procedures recommended by the manufacturer.

Alternatively, chemical activation, of the hydroxyl-group-containingassay matrix (cellulose paper or membrane) can be performed byincubating a 20×25 cm sheet of membrane or paper in a covered bakingdish 23×28 cm for 2; bouts at room temperature in 500 mL of 0.2 M1,1′-carbonyldiimidazole (CDI, Aldrich product no 11, 553-31. Followingthis incubation, the activated membrane is washed extensively in several(4-8) 250 mL volutes of methylene chloride and dried under nitrogen.This procedure results in activated membrane to which proteins or smallmolecules with primary amine functional groups can be covalentlyimmobilized (non-diffusive binding). Non-diffusive-immobilization of theprotein to the activated membrane, either prepared at outlined above orusing ore of the commercial activated membranes, is accomplished byincubating the activated membrane in 100 m. of a 0.01 mg/mL to 10 mg/mLsolution of the desired immobilization compound in 0.1 M sodiumphosphate, pH 7, at room temperature for two hours. The paper is thenwashed by incubation for 20 Minutes in 500 mL of 0.1 M sodium phosphatepH 7. The washing step is repeated 4 times, then the paper is soaked in150 mL of 0.5% polyvinyl alcohol (PVA, Aldrich 18, 965-0) for 10minutes, gently blotted and dried in a convection oven at 45° C. for 10to 30 minutes or until dry.

Colloidal gold conjugates of mouse IgG, 1H11 (monoclonal anti-NTx), andNTx are commercially available from EY Laboratories, San Mateo, Calif.Methods for the preparation, of colloidal gold conjugates are disclosedin Muller, C., et al., J. Imm. Methods, 37, 185-190 (1980); Roth, J.,“Techniques in Immunocytochemistry,” Academic Press, pp. 219-284.Conjugates include: Gold-1H11 (15 nm particle, monoclonal anti-NTx);Gold-Mouse IgG1 Kappa (15 nM particle); Gold-NTx (15 nm particles).

Preparation of Latex Particle Conjugates Accomplished by immobilizingNTx, NTx-protein conjugate, MAb-1H11 or Mouse IgG to 0.356 μmmicrospheres (Bangs stock #D0003561CB) to produce. NTx-Latex,NTx-protein-latex and MAb 1H11-Latex conjugates. To a suspension of0.356 μm carboxylated microspheres add 10 molar equivalents (relative tothe COOH groups on the bead surface) of 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDAC, Sigma E 0388) and 10 molar equivalents ofN-Hydroxysuccinimide (NHS, Pierce 24500) in 0.1 M sodium phosphate, pH7.0, and 0.5% Tween 20 at room temperature with stirring for 3.0minutes. Purify by centrifugation at 13,000 RPM, for 15 minutes.followed by washing with 10 mM sodium phosphate pH 7, 0.5% Tween 20. Addeither the NTx, NTx-protein conjugate, or MAb-1H11 at a ten fold molarexcess to a stirring solution of the activated microspheres in 0.1 Msodium phosphate, pH 7.0, 0.5% Tween 20, and allow to react for 2 hoursat room temperature; then purify by centrifugation with washing anddialysis. The microparticles now have the desired immobilizationcompound non-diffusively immobilized.

Assay strips and reagents were prepared as discussed in the first strippreparation method and the first non-diffusive immobilization method.above. The strips were either 0.5 cm wide by 6 cm long or 0.3 cm wide,by 3 cm long, depending on the experiment. The following assay protocolwas used:

-   1. Sample was added (25-100 μL) to the bottom, of. a 12×7.5 mm. test    tube.-   2. An assay strip (reagents already applied) was inserted into the    test tube. Sufficient time, was allowed for wicking to completely    saturate the strip. This required about 5-8 minutes for the 0.5 cm×6    cm strips and about 1-3 minutes for the 0.3 cm by 3 cm strips.-   3. The test zone on the assay strips were read with a Gretag model    D182 reflectance densitometer.

Example 2

This example demonstrates a single-step, quantitative, lateral flow,inhibition type immunoassay for a small molecule using colored particlesas the detection method. The assay summarized below in Table 1 wasconducted using reagents and methods as described in Example 1.

The assay strips of this example were 6 cm wide and 0.5 cm long and weresimilar to those shown in FIG. 1 and FIG. 3, with the exception that thethird zone 18 (second test zone) was not included. The immunoassay stripconfiguration was as follows:

-   -   (1) a lower 8 μm pore size nitrocellulose section containing a        reagent zone 14 having diffusively applied MAb-1H11-colloidal        gold, prepared as described above;    -   (2) a 0.5 cm test zone 16 containing non-diffusively immobilized        NTx covalently linked via the native primary amine group to Pall        IMMUNODYNE® ABC, prepared as described above; and    -   (3) an upper wick area of 8 μm pore size nitrocellulase that        extended from the upper edge of zone 16 to the top of the strip.

All strip zones were laminated in physical communication with adjacentzone (s), permitting fluid to flow through the entire strip by wickingaction, and were supported on a plastic backing as outlined above.

NTx was diluted in PBS to the concentrations indicated in Table 1 below,and the general assay protocol of Example 1 was used to generate thedata shown in Table 1. These data indicate a dose-response demonstratinggood sensitivity and quantitative performance for the present invention.The assay results in Table 1 demonstrate that the present assay stripand method can distinguish a 1 nM concentration of the peptide markerNTx from the background (0 concentration) using colloidal gold as thesignal reagent.

TABLE 1 NTx Dose Response Conjugate: Colloidal Gold-1H11 ReflectanceDensity NTx (nM) (Gretag) 0 0.60 1 0.57 30 0.55 100 0.52 300 0.41 10000.21 3000 0.18

Example 3

This example demonstrates a quantitative, lateral flow, inhibition typo,immunoassay which shows excellent performance using colored particles atthe indicator. The assay summarized below in Table 2 was conducted usingstrips, reagents and methods as described in Example 2.

The assay protocol as indicated above in Example 1 was used to generatedata in the non-amplified data. column of Table 2. For the silveramplified assays, the protocol was, followed as in Example 1, with theexception, that a silver enhancement reagent was added to the test zoneafter the colloidal gold binding. The results in Table 2 demonstrate adose-response showing excellent. sensitivity and quantitativeperformance, for the present invention.

TABLE 2 NTx Dose Response Silver Amplification Conjugate:Colloidal-Gold-1H11 Reflectance Density Gretag NTx Non- Silver- (nM)Amplified Amplified 1 0.30 1.07 30 0.25 0.91 100 0.19 0.84 300 0.15 0.591000 0.09 0.23 3000 0.08 0.02

Example 4

This example, demonstrates a single-step, quantitative, lateral flow,competitive type immunoassay for a protein using colored particles asthe indicator. The assay summarized below. in Table 3 was conductedusing reagents and methods as described in Example 1.

The assay strips of thia example were 6 cm wide and 0.5 cm long and weresimilar to those shown in FIG. 1 and FIG. 31, with the exception thatthe first test zone (second. zone 16) was not included. The immunoassaystrip configuration was as follows:

-   -   (1) a lower 8 μm pore size nitrocellulose section containing        continuous reagent zones 14 and 16, zone 14 having diffusively        applied mouse IgG-colloidal gold, prepared as described above;    -   (2) a 0.5 cm test zone 18 containing non-diffusively immobilized        goat anti-mouse adsorbed to 8 μm pore size nitrocellulose,        prepared as described above; and    -   (3) an upper wick area of 8 μm, pore size nitrocellulose that        extended from the upper edge of zone 18 to the top of the strip.

All strip zones were laminated in physical communication with adjacentzone(s), permitting fluid to flow through the entire strip by wickingaction, and were supported on a plastic. backing as outlined above.

Mouse IgG was diluted in PBS to the concentrations indicated in Table 3below, and the assay protocol of Example 1 was used to generate the datashown in Table 3. These data. demonstrate a dose-response showing goodsensitivity and quantitative performance for the present invention.

TABLE 3 IgG Dose Response Conjugate: Colloidal Gold-IgG1 ReflectanceMouse IgG Density (μg/mL) (Gretag) 0 0.13 8 0.10 33 0.09 50 0.08 1000.06 200 0.05

Example 5

This example demonstrates a single-step, quantitative, lateral flow,inhibition type immunoassay for a small molecule using colored particlesas the indicator and an assay reference that is directly related to theassay function. The assay summarized below in Table 4 was conductedusing reagents and methods described, in Example 1

The assay strips of this example were 6 cm wide and 0.5 cm long and axeshown in FIG. 1 and FIG. 3. The immunoassay strip configuration was asfollows:

-   -   (1) a lower 8 μm pore size nitrocellulose section containing a        reagent zone 14 having diffusively applied MAb-1H11-colloidal        gold, prepared as described above;    -   (2) a (1.5 cm test zone 16 containing non-diffusively        immobilized NTx covalently linked via the native primary amine        group to Pall IMMUNODYNE® ABC, prepared as described above;    -   (3) a 0.5 cm spacer 22 of 8 μm pore size nitrocellulose;    -   (4) a 0.5 cm long test zone 18 of 8 μm pore size mitrocellulose        containing non-diffusively immobilized, adsorbed        goat-anti-mouse; and    -   (5) an upper wick area of 8 μm pore size nitrocellulose that        extended from the upper edge of zone 18 to the top of the strip.

All strip zones were laminated in physical communication with adjacentzone(s), permitting fluid to flow through, the entire strip by wickingaction, and were supported on a plastic lacking as outlined above.

NTx was diluted in PBS to the concentrations indicated in Table 4 below,and the general assay protocol of Example 1 was used to generate thedata shown in Table 4. These data demonstrate a dose-response in bothtest zone 16 and test zone 18 showing good sensitivity and quantitativeperformance for the present invention. The sum of the two test zonesremained substantially constant, this indicating, a correct functioningof the internal reference feature of the present invention, wherein thesum of the signal from test zone one and test one two provides areliable quality reference to assure correct assay performance.

TABLE 4 NTx Dose Response Two Test Zones Conjugate: Colloidal Gold-1H11Reflectance Density (Gretag) NTx (nM) Test Zone 1 Test Zone 2 Sum 1 0.380.10 0.48 30 0.32 0.16 0.48 100 0.21 0.25 0.46 300 0.14 0.35 0.49 10000.09 0.40 0.49 3000 0.07 0.47 0.54

Example 6

This example demonstrates a quantitative, lateral flow, inhibition typeimmunoassay for a small molecule using an enzyme label as the detectionsystem. The assay summarized, below in Table 5 was conducted usingstrips, reagents and methods as described in Example 2, with theexception that the lower reagent zone 14 contained, diffusively appliedHRP-1H11. Five pi, of 0.1 μg/mL HRP-1H11 was applied to zone 14. Asindicated in table 5, an approximately 1.5 ing/mL solution of NT-IgGconjugate was diluted 1:10, 1:20 and 1:40, the adsorbed as described inExample 1 to the 8 μm pore size nitrocellulose of test zone 16.

NTx was diluted in PBS to the concentrations indicated in Table 5 below,and the assay protocol of Example 1 was used to generate the data shownin Table 5. These data illustrate dose-responses at each of threedifferent NTx immobilization conditions which show good sensitivity andquantitative performance. for the present invention when an enzymeindicator is used.

TABLE 5 NTx Dose Response Conjugate HRP-1H11 at 0.1 μg/mL VariousNTx-protein immobilization concentrations NTx-Protein Dilution NTx 1:101:20 1:40 (nM) Reflectance Density (Gretag) 24 0.47 0.49 0.52 80 0.410.40 0.42 240 0.32 0.38 0.30 800 0.27 0.27 0.20 2400 0.16 0.19 0.17

Example 7

This example demonstrates a single-step, quantitative, lateral flow;competition-type immunoassay for a small molecule using coloredparticles as the indicator. The assay summarized below in Table 6 wasconducted using reagents and methods as described in Example 1.

The assay strips of this example were 3 cm wide and 0.3 cm long, andwere similar to those shown in FIG. 1 and FIG. 3, with the exceptionthat the third zone 18 (second test zone) was not included. Theimmunoassay strip configuration was as follows:

-   -   (1) a lower 8 μm pore size nitrocellulose section containing        continuous reagent zone 14 having diffusively immobilized.        NTx-latex beads (blue), prepared as described above;    -   (2) a 0.3 cm test zone 18 containing non-diffusively immobilized        1H11 monoclonal anti-NTx, adsorbed to 8 μm pore size        nitrocellulose, prepared as described above; and    -   (3) an upper wick area of 8 μm pore size nitrocellulose that        extended from the upper edge of zone 16 to the top of the strip.

All strip zones were laminated in physical communication with adjacentzone(s), permitting fluid to flow through the entire strip by wickingaction, and were supported on a plastic backing as outlined above.

NTx was diluted in PBS to the concentrations indicated in Table 6 below,and the general assay protocol of Example 1 was used to generate thedata shown in Table 6. These data illustrate a dose-responsedemonstrating good sensitivity and quantitative performance for thepresent invention using blue latex particles as the signal reagent.

TABLE 6 NTx Dose Response Conjugate: Latex Bead-NTx 1H11 Immobilized NTxReflectance (nM BCE) Density 30 0.79 100 0.64 300 0.46 1000 0.33 30000.20

Example 8

This example demonstrates a single-step, quantitative, lateral flow,competition-type immunoassay for a proteins using colored particles asthe indicator. The assay summarized below in Table 7 was conducted usingreagents and methods as described In Example 1. The assay strips of thisexample were 3 cm wide and 0.3 cm long, and were similar to those shownin FIG. 1 and FIG. 3, with the exception that the second zone 16 (firsttest zone) was not included. The immunoassay strip configuration was asfollows:

-   -   (1), a lower 8 μm pore size nitrocellulose section, containing        reagent zone 14 having diffusively applied mouse IgG-latex beads        (blue);    -   (2) a 0.3 cm intermediate section, of 8 μm pore size        nitrocellulose, containing test zone 18 having non-diffusively        immobilized goat anti-mouse adsorbed thereto; and    -   (3) an upper wick area of 8 μm pore size nitrocellulose that        extended from the upper edge of zone 18 to the top of the strip.

All strip zones were laminated in physical communication with adjacentzone(s), permitting fluid to flow through the entire strip by wickingaction, and were supported on a plastic backing as outlined above.

Mouse IgG was diluted in PBS to the concentrations indicated. in Table 7below, and the general assay protocol of Example 1 was used to generatethe data shown in Table 7. These data illustrate a dose-responsedemonstrating good sensitivity and quantitative performance for thepresent invention using blue latex particle as the signal reagent.

TABLE 7 IgG Dose Response Conjugate: NTx-IgG-Latex Beads Goat anti-mouseimmobilized Mouse IgG Reflectance (μg/mL) Density 0 0.62 4 0.52 8 0.4433 0.37 50 0.33 100 0.26 200 0.23

Example 9

This example demonstrates a single-step, quantitative, lateral flow,competitive-type immunoassay for a small molecule and a large molecule,using, colored particles as the indicator and an assay reference that isdirectly related to the assay function. The assay summarized below inTable was conducted. using reagents and methods as described inExample 1. The assay strips of this example were 3 cm wide and 0.3 cmlong, and are shown in FIG. 1 and FIG. 3. The immunoassay stripconfiguration was as follows:

-   -   (1) a lower 8 μm pore size nitrocellulose section, containing        reagent zone 14 having diffusively immobilized NTx-latex beads        10.412 μm, blue);    -   (2) a 0.3 cm intermediate section of 8 μm pore size        nitrocellulose, containing test zone 18 having non-diffusively        immobilized 1H11 monoclonal anti-NTx adsorbed thereto;    -   (3) a 0.5 cm spacer of 8 μm pore size. nitrocellulose;    -   (4) a 0.3 cm long section of 8 μm pore size nitrocellulose,        containing, test zone 18 having non-diffusively immobilized        goat-anti mouse adsorbed thereto; and    -   (5) an upper wick area of 8 μm pore size nitrocellulose that        extended from the upper edge. of zone. 18 to the top of the        strip.

All strip zones were laminated in fluid communication with adjacentzone(s), permitting fluid. to flow through the entire strip by wickingaction, and were supported on a plastic backing as outlined above.

NTx was diluted in PBS to the concentrations indicated in Table 8, andthe general, assay protocol of Example 1 was used to generate the datashown in Table 8. These data illustrate a dose-response in both testzone 16 and test zone 18 demonstrating good sensitivity and quantitativeperformance for the present invention. The sum of the signals from thetwo test zones remains substantially constant throughout the NTXconcentration, range, indicating a correct functioning, of the assay.

According to the present invention, the sum of the signals from testzones 16 and 18 provides an internal quality reference to assurereliable and, correct. assay performance. In addition, better assaysensitivity is seen in test zone 18 at the lower end of the curve (0 to30 nM NTx), while better separation (Sensitivity) is seen. in test zone.16 at the upper end of the curve (100 to 300 nM NTx). These resultssuggest a that hybrid calibration algorithm using both test zones canprovide improved performance, relative to previously de-Scribedsingle-test-zone methods. According to the present invention, assaycalibration and instrumental reading can use a single test zone alone,or both test zones dither separately or combined, to provide maximumsensitivity and reliability.

TABLE 8 NTx Dose Response Two Test Zones Conjugate: Latex Bead-NTxImmobilized Zone 1-1H11, Zone 2-Goat Anti-Mouse Reflectance Density(Gretag) NTx (nM) Zone 1 Zone 2 Sum 0 1.01 0.23 1.24 1 1.06 0.21 1.27 301.05 0.40 1.45 100 0.96 0.50 1.46 300 0.68 0.57 1.25

Example 10

The assay summarized below in Table 9 was conducted using strips,reagents. and methods as described in Example 9, with the exception thatmonoclonal rat anti-mouse was immobilized in zone 18.

The conclusions of Example 9 are supported by these data.

TABLE 9 NTx Dose Response Two Test Zones Conjugate: Latex Bead-NTxImmobilized Zone 11H11, Zone 2 Monoclonal Rat-Anti-Mouse ReflectanceDensity (Gretag) NTx (nM) Zone 1 Zone 2 Sum 0 0.99 0.37 1.36 1 0.86 0.401.26 30 0.84 0.44 1.28 100 0.79 0.56 1.35 300 0.38 0.62 1.00

Example 11

The test results graphically represented in FIG. 8 were from an assayconducted using strips, reagents and methods as described in Example 9,except as otherwise noted. FIG. 8 illustrates the NTx dose responseusing IgG-C-peptide in the first test zone on nitrocellulose membrane,S&S AE 98, with a pore size of 5 μm. The second-test zone immobilizedmonoclonal rat anti-mouse on nitrocellulose.

Numerous modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described herein.

1-50. (canceled)
 51. A device for determining the presence of an analytein a sample, the device comprising: a bibulous member capable of beingtraversed by the sample; a first zone on the bibulous member forreceiving and contacting the sample containing a diffusivelyimmobilized, particle-linked antigen, the particle-linked antigenreacting in the presence of the analyte to form a mixture; a second zoneon the bibulous member for receiving and contacting the mixturecontaining a non-diffusely immobilized antibody capable of binding theparticle-linked antigen and free sample antigen, the second zoneexpresses a detectable response inversely related to the analyte levelin the sample; a third zone on the bibulous member for receiving andcontacting the remaining mixture containing a non-diffusivelyimmobilized first member of a specific binding pair, capable ofspecifically binding to its specific binding partner which is the secondmember of the specific binding pair on the surface of theparticle-linked antigen, which second member of the specific bindingpair is not antigenically related to the sample antigen and will noteffectively compete with the antigen to bind to an anti-antigenmonoclonal antibody, the third zone expresses a detectable responserelated to the analyte level in the sample; and means for determiningthe analyte level in the sample from the detectable responses in thesecond and third zones.
 52. The device of claim 51 wherein thedetermining means further comprises determining the sum of thedetectable responses from the test zones to be within a pre-determinedrange of response.
 53. The device of claim 51 wherein the determiningmeans further comprises determining the level of analyte in one testzone by comparison to the total detectable response.
 54. The device ofclaim 51 wherein the determining means further comprises determining thelevel of analyte using the detectable response in one test zone bycomparison to a pre-determined standard.
 55. The device of claim 51wherein the determining means further comprises determining the level ofanalyte using the detectable response in one test zone in comparison tothe detectable response in the other test zone.
 56. The device of claim51 wherein the determining means further comprises determining the levelof analyte using the detectable response in one test zone in one portionof the pre-determined range of analyte and the detectable response inthe other test zone in a different portion of the pre-determined rangeof analyte.
 57. A device for determining the presence of an analyte in asample, the device comprising: a bibulous member capable of beingtraversed by the sample; a first zone on the bibulous member forreceiving and contacting the sample containing a diffusively immobilizedparticle-linked antibody, the particle-linked antibody reacting in thepresence of the analyte to form a conjugate included in a mixture; asecond zone on the bibulous member for receiving and contacting themixture containing a non-diffusively immobilized antigen capable ofbeing bound by the particle-linked antibody, the second zone expresses adetectable response inversely related to the analyte level in thesample; a third zone on the bibulous member for receiving and contactingthe remaining mixture containing a non-diffusively immobilized firstmember of a specific binding pair capable of specifically binding to itsspecific binding partner which is the second member of the specificbinding pair on the surface of the particle-linked antigen, which secondmember of the specific binding pair is not antigenically related to thesample antigen so it will not effectively compete with the antigen tobind to an anti-antigen monoclonal antibody, the third zone expresses adetectable response related to the analyte level in the sample; andmeans for determining the analyte level in the sample from thedetectable responses in the second and third zones.
 58. The device ofclaim 57 wherein the determining means further comprises determining thesum of the detectable responses from the test zones is within apre-determined range of response.
 59. The device of claim 57 wherein thecombining means further comprises determining the level of analyte inone zone by comparison to the total detectable response.
 60. The deviceof claim 57 wherein the combining means further comprises determiningthe level of analyte using the detectable response in one test zone bycomparison to a pre-determined standard.
 61. The device of claim 57wherein the combining means further comprises determining the level ofanalyte using the detectable response in one test zone in comparison tothe detectable response in the other test zone.
 62. The device of claim57 wherein the combining means further comprises determining the levelof analyte using the detectable response in one test zone in one portionof the pre-determined range of analyte and the detectable response inthe other test zone in a different portion of the pre-determined rangeof analyte.
 63. A method for determining the level of at least oneanalyte in a sample, the method comprising the steps of; contacting thesample with an end portion of a bibulous strip having a plurality ofzones; wicking the sample to a labeled indicator reagent diffusivelyimmobilized on the bibulous strip; reacting the labeled indicatorreagent in the presence of the analyte to form a mixture; wicking themixture to a first reagent non-diffusely immobilized on the bibulousstrip; reacting the first reagent in the presence of the mixture to forma first reaction product and a detectable response inversely related tothe analyte level in the sample; wicking the remaining mixture to asecond reagent non-diffusely immobilized on the bibulous strip; reactingthe second reagent in the presence of the remaining mixture to form asecond reaction product and a detectable response related to the analytelevel in the sample; and determining the analyte level in the samplefrom the detectable responses in the reacting steps with the first andsecond reagents.
 64. The method of claim 63 wherein: the reacting stepwith the labeled indicator reagent comprises forming a mixture includingparticle-linked antigen with the analyte; the reacting step with thefirst reagent comprises binding an antibody with the particle-linkedantigen and the analyte; the reacting step with the second reagentcomprises binding a first member of a specific binding pair to a secondmember of the specific binding pair on the particle-linked antigen, thesecond member of the specific binding pair is not a specific bindingpartner to the analyte.
 65. The method of claim 63 wherein: the reactingstep with the labeled indicator reagent comprises forming a mixtureincluding a particle-linked antibody with the analyte; the reacting stepwith the first reagent comprises substantially binding theparticle-linked antibody with an immobilized antigen; the reacting stepfor the second reagent comprises binding a first member of a specificbinding pair to a second member of the specific binding pair on theparticle-linked antibody, the second member of the specific binding pairis not a specific binding pair to the analyte.